Project Summary Glycyl radical enzymes (GREs) catalyze diverse chemical transformations critical to both human health and the environment. The unifying characteristic of GREs?a stable glycyl radical cofactor?enables challenging, otherwise inaccessible chemistry, but is extremely oxygen sensitive. Thus, this still expanding family of enzymes is prevalent in anaerobic environments such as the human gut and marine seeps. GRE activity has been implicated in liver, heart, and kidney diseases and could prove uniquely effective in bioremediation efforts; however, most GREs remain uncharacterized. Additionally, the molecular detail of how the large breadth of GRE-catalyzed chemistry is accomplished is not yet understood. The work described within this proposal will further illuminate GRE mechanism as well as explore GRE use in biocatalysis. Specifically, this work will explore a class of GREs termed X-succinate synthases (XSS). XSS enzymes catalyze the hydroalkylation of fumarate, in which new C?C bonds are forged between fumarate and unactivated hydrocarbon substrates. Through this mechanism, XSS-containing organisms are able to degrade hydrocarbon pollutants in even the most recalcitrant regions for environmental remediation. Beyond bioremediation, XSS enzymes enable remarkable chemistry and could serve as an important addition to our current C?H functionalization toolkit. Here, I aim to understand XSS enzymes in molecular detail though structural biology and develop novel methodologies that will allow for engineering of this enzyme class, as well as radical enzymes more broadly. Collectively, this work will allow us further insight into the ways in which Nature uses enzymes to achieve remarkable chemistry and will allow us to begin to harness the powerful radical chemistry Nature has to offer.